1. Field of the Invention
The present invention-relates to capsules, in particular microcapsules, comprising a polyanionic bead core and a polycationic membrane, and to a process for their production. More especially, the invention relates to such capsules having a higher strength and to a process for their formation.
2. Description of Related Art
Capsules consisting of a gelled polyanionic core and a polycationic membrane are known in the art and examples have comprised a core of an alginate and a membrane layer of poly-L-lysine. Capsules having a membrane layer comprising chitosan are also known. Such capsules have found various uses, such as the encapsulation of cells or other biological matter, (as described in "Alginate as immobilisation matrix for cells", Olav Smidsro/d, Gudmund Skjak-Braek, TIBTECH, March (1990), Vol. 8, No. 3[74]), in drug delivery, especially sustained release drug delivery, and also have other agricultural and industrial uses.
Alginates are salts of alginic acids which are linear polysaccharides consisting of (1-4) bound residues of .beta.-D-mannuronic acid ("M units") and .alpha.-L-guluronic acid ("G units"). Alginate polymers may consist of homopolymeric sequences of mannuronic acid residues ("M blocks"), homopolymeric sequences of guluronic acid residues ("G blocks") and sequences including both mannuronic and guluronic acid residues ("MG" blocks). Alginates will usually contain all three types of block each comprising from about three to about twenty monomer units. The distribution and relative quantities of the M and G units influences the properties of the alginate, and depends on the source of the alginate. Alginates are most commonly extracted from various types of algae.
Alginate gels can be formed by cross-linking the alginate polymer units and suitable cross-linking agents are polyvalent or divalent (hereinafter polyvalent) cations, in particular Ca.sup.2+ and A1.sup.3+.
A particular advantage, especially in relation to biological matter, of capsules prepared from polysaccharides, such as alginates, is that the encapsulation can be performed under mild conditions. Thus, gelling of alginate can take place at room temperature and in aqueous conditions. The procedure is rapid and does not require organic cross-linking agents or solvents. Alginates also have the particular advantage of being non-toxic and suitable for use in food and as pharmaceutical adjuvants or excipients.
Although alginate beads per se have been considered for uses such as sustained drug delivery, alginates have significant disadvantages in terms of stability and porosity. Alginates are biodegradable and alginate gels are reversible ionic networks which can be destabilised by calcium sequestering agents, such as citrates and phosphates, and also by non-gelling ions, such as Na.sup.+ and Mg.sup.2+. Also, the pore structure of the alginate gel is unsuitable for sustained drug delivery. For this reason, capsules have been prepared by creating a membrane on the surface of the alginate bead. Most commonly, the membrane has been formed from the polycation poly-L-lysine, but chitosan (also a polycation) has also been used. In this way, a polycationic barrier is formed around the alginate gel which provides added stability and which can be effective in controlling parameters, such as pore size and the rate of drug release in sustained drug delivery.
Capsules of the above general type, methods of making them and their uses are known in the art and are described in, for example, the following patents:
U.S. Pat. No. 4,352,883 discloses a basic process for encapsulating a core material, such as a viable cell, within capsules having semipermeable membranes. This process comprises suspending the core material in a solution of a water-soluble polyanionic polymer capable of being gelled (especially an alginate salt), forming droplets which are then suspended in a solution of a polyvalent cation, such as Ca.sup.2+, and thereby producing soft, shape-retaining, hydrated gelled masses. Thereafter, a membrane is formed about each of these gelled masses by reaction of the anionic groups from the polyanionic polymer hydrogel with cationic groups from a polycationic polymer. Useful polymeric cations include proteins and aminated polysaccharides or aminated polymers. The most preferred polycationic of this reference is polylysine.
Porosity control is an important factor in a number of important uses of such capsules. For example, the capsule membrane can be used for differential screening to separate molecules on a weight basis. U.S. Pat. No. 4,409,331 discloses a method of differential screening wherein lower molecular weight molecules secreted by a cell within the capsule core may traverse the capsule membrane while other higher molecular weight molecules are confined within the capsule. This is achieved through control of permeability within limits by selecting the molecular weight of the cross-linking polymer used and by regulating the concentration of the polymer solution and the duration of exposure. In general, the higher the molecular weight of the polymer solution and the less penetration, the larger the pore size.
In U.S. Pat. No. 4,690,682 it is further disclosed that release of the core material may be achieved through reliquifying the intracapsular volume by immersing the capsule in a solution of a sequestering agent that will remove the multivalent gelforming ions from the network. In this case, only the pore size of the membrane will have a determining effect on the sustained release properties of the capsule. However, in order to sustain the necessary osmotic gradient, a very large reservoir of core material must be maintained within the capsule and the core material which is released must be removed from the exterior of the capsule at a relatively rapid rate.
From U.S. Pat. No. 4,749,620, it is known to prepare polymer complex capsules having a liquid core through the direct formation of capsules from two solutions of polymers, where a polyanionic polymer solution is added dropwise to a polycationic polymer solution or vice versa. This is the so-called "one-step" process. Unfortunately, capsules having a liquid core show a lack of strength, which may make them unacceptable especially in uses where a certain capsule life time is necessary.
From U.S. Pat. No. 4,663,286 it is known to improve membrane uniformity and porosity control by expanding the gelled alginate beads in a saline bath essentially free from polyvalent cations, to remove some of the polyvalent cations from the alginate and further hydrate said gelled masses, and finally to form a membrane about the hydrated gelled masses to form capsules by reaction between the anionic groups on the alkali metal alginate and cationic groups on a polycationic polymer. Optionally, further membrane layers can be formed by either coating the membrane layer with an anionic polymer or a cationic polymer.
While poly-L-lysine (hereinafter polylysine) has most commonly been used for the formation of the membrane layer of the capsules, it has particular disadvantages in use. Thus, polylysine is known to be an irritant which makes it less suitable for use in pharmaceutical preparations, more especially for preparations intended for use in easily irritated areas such as the throat or stomach. Also, it has been suggested that polylysine can be enzymatically degraded which in some applications will reduce unacceptably the life time of the capsule. The prior art has addressed this problem by adding further layers of polyanionic or polycationic polymers, but this is expensive and cumbersome. It may also be noted the polylysine itself is an expensive synthetic protein.
As an alternative to polylysine, chitosan has been proposed because it is a natural, non-toxic, biodegradable polymer already known as a natural component in many consumer food products and is also cheaper than polylysine. Chitosan is a partially de-acetylated chitin, which is one of the most common biopolymers in nature and which appears in many organisms as a structural component. Through the deacetylation of chitin (which is insoluble) chitosan may be produced which is soluble in acidic solutions. Commercially available chitosans generally contain about 75 to 95% glucosamine units and about 5 to 25% N-acetylglucosamine units connected through (1-4) linked .beta.-glycosidic linkages. Through the amino groups, chitosan may be protonated with the result that chitosan is one of the few biopolymers which are cations at physiological pH.
However, chitosan has not found acceptance because capsules prepared by the addition of cross-linked polyanionic (e.g. alginate) beads to a chitosan solution have been found to have insufficient strength. This is because it has been possible to provide only a small, thin layer of the chitosan on the polyanionic polymer (e.g. alginate) bead. Commercially available chitosans which have been used in prior art capsules have had a high molecular weight of 100,000 or more. The reaction time required to form a layer of chitosan on an alginate bead is also considerably longer than the time required to provide a corresponding polylysine layer.
In view of the above, the present invention seeks to provide a method of preparing polyanion-polycation (e.g. alginate-chitosan) capsules of higher strength.